Protein stabilized measurement methods

As described in the following chapter, there are many biopharmaceutical applications of protein assays. Assigning the protein concentration for the drug substance, drug product, or in-process sample is often the first task for subsequent analytical procedures because assays for purity, potency, or identity require that the protein concentration be known. Hence it is typical for several different methods to be employed under the umbrella of protein concentration measurement, depending on the requirements of speed, selectivity, or throughput. The protein concentration is valuable as a stand-alone measurement for QC and stability of a protein. However, protein concentration methods provide no valuable... 

Powell, K.D. Wales, T.E. Fitzgerald, M.C. Thermodynamic stability measurements on multimeric proteins using a new H/D exchange-and matrix-assisted laser desorption/ ionization (MALDl) mass spectrometry-based method. Protein Sci. 2002, 11, 841-851. 

ProTherm (16) is a large collection of thermodynamic data on protein stability, which has information on 1) protein sequence and stmcture (2) mutation details (wild-type and mutant amino acid hydrophobic to polar, charged to hydrophobic, aliphatic to aromatic, etc.), 3) thermodynamic data obtained from thermal and chemical denaturation experiments (free energy change, transition temperature, enthalpy change, heat capacity change, etc.), 4) experimental methods and conditions (pH, temperature, buffer and ions, measurement and method, etc.), 5) functionality (enzyme activity, binding constants, etc.), and 6) literature. 

The influence of various cosolvents on protein stability has been discussed by Timasheff [50]. There has been a considerable debate in the literature on the number of water molecules that are taking part in protein-protein or protein-DNA interactions as estimated by various methods. A recent theoretical analysis suggests that the osmotic stress method may overestimate the number of waters involved [51]. These models assume that the cavities that are formed at the interface between macromolecules do not contribute to the measured volume changes as suggested by Silva and Weber [31]. 

Wang and Johnson (2001) reported on test measurement methods that were major indicators of soybean oil quality. These tests included peroxide value, anisidine value, FFA content, phospholipid content, total tocopherol content, oxidative stability index, color, and moisture content. For soybean meal, they reported on urease activity, protein dispersibility index (PDI), rumen bypass or rumen undegradable protein, trypsin inhibitor activity, moisture content, residual oil content, protein content, fiber content, color, amino acid profiles, and protein solubility under alkaline (KOH) conditions. 

The dynamic foam stability is usually measured by the volume of foam at a specific equilibrium flow rate, while the static foam stability is measured by the rate of collapse. Dynamic measurements are particular relevant for transient foams, while for foams of high stability, the static or equilibrium methods are usually more useful, particular for highly stabilized foams such as protein-stabilized foam systems. 

In tissues, most coelenterazine exists in a protein-bound stabilized form, which liberates free coelenterazine when extracted with methanol. Thus, the amount of coelenterazine measured by this method is the sum of free coelenterazine and its protein-bound form. 

D. Similar methods were used for modification of the enzymes listed in Table II as well as bovine hemoglobin (see Table III). The choice of conditions for the modification reactions (pH, temperature, etc.) was made mainly based on the properties/stability of each protein. Enzymatic activities were measured by previously reported methods (77,27-25). 

Determine the minimum amount of protein A required to stabilize the colloidal gold sol being used. The colloidal suspension should be adjusted, if needed, with 0.1M K2CO3 to pH 6-7. Measure the pH of the sol using a gel-filled electrode. Determining the stabilization amount of protein A can be done according to the method described in Section 1, this chapter. 

---